Establishment of two serological methods for detecting IgG and neutralizing antibodies against Crimean-Congo hemorrhagic fever virus glycoprotein.

Introduction: The Crimean-Congo hemorrhagic fever virus (CCHFV), the most geographically widespread tick-borne virus, is endemic in Africa, Eastern Europe and Asia, with infection resulting in mortality in up to 30% of cases. Currently, there are no approved vaccines or effective therapies available for CCHF. The CCHFV should only be manipulated in the BSL-4 laboratory, which has severely hampered basic seroprevalence studies. Methods: In the present study, two antibody detection methods in the forms of an enzyme-linked immunosorbent assay (ELISA) and a surrogate virus neutralization test (sPVNT) were developed using a recombinant glycoprotein (rGP) and a vesicular stomatitis virus (VSV)-based virus bearing the CCHFV recombinant glycoprotein (rVSV/CCHFV) in a biosafety level 2 (BSL-2) laboratory, respectively. Results: The rGP-based ELISA and rVSV/CCHFV-based sVNT were established by using the anti-CCHFV pre-GC mAb 11E7, known as a broadly cross-reactive, potently neutralizing antibody, and their applications as diagnostic antigens were validated for the specific detection of CCHFV IgG and neutralizing antibodies in experimental animals. In two tests, mAb clone 11E7 (diluted at 1:163840 or 512) still displayed positive binding and neutralization, and the presence of antibodies (IgG and neutralizing) against the rGP and rVSV/CCHFV was also determined in the sera from the experimental animals. Both mAb 11E7 and animal sera showed a high reactivity to both antigens, indicating that bacterially expressed rGP and rVSV/CCHFV have good immunoreactivity. Apart from establishing two serological testing methods, their results also demonstrated an imperfect correlation between IgG and neutralizing antibodies. Discussion: Within this limited number of samples, the rGP and rVSV/CCHFV could be safe and convenient tools with significant potential for research on specific antibodies and serological samples.

Host response transcriptomic analysis of Crimean-Congo hemorrhagic fever pathogenesis in the cynomolgus macaque model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne RNA virus prevalent in Asia, Europe, and Africa, and can cause a hemorrhagic disease (CCHF) in humans with mortality rates as high as 60%. A general lack of both effective medical countermeasures and a comprehensive understanding of disease pathogenesis is partly driven by an historical lack of viable CCHF animal models. Recently, a cynomolgous macaque model of CCHF disease was developed. Here, we document the targeted transcriptomic response of non-human primates (NHP) to two different CCHFV strains; Afghan09-2990 and Kosova Hoti that both yielded a mild CCHF disease state. We utilized a targeted gene panel to elucidate the transcriptomic changes occurring in NHP whole blood during CCHFV infection; a first for any primate species. We show numerous upregulated genes starting at 1 day post-challenge through 14 days post-challenge. Early gene changes fell predominantly in the interferon stimulated gene family with later gene changes coinciding with an adaptive immune response to the virus. There are subtle differences between viral strains, namely duration of the differentially expressed gene response and biological pathways enriched. After recovery, NHPs showed no lasting transcriptomic changes at the end of sample collection.

Sequential determination of viral load, humoral responses and phylogenetic analysis in fatal and non-fatal cases of Crimean-Congo hemorrhagic fever patients from Gujarat, India, 2019.

BACKGROUND: Thirty-four CCHF cases (17 fatal; 17 survived) were confirmed from Gujarat state, India during the year 2019. We aimed to find out the viral load, antibody kinetics, cytokine profile and phylogenetic analysis between fatal and non- fatal cases. METHODS: Thirty four cases were included in this study. Blood and urine samples were collected from all the cases on the day of admission to hospital. Non-fatal cases were followed weekly for understanding the profile of viral kinetics, anti-CCHFV IgM and IgG antibodies. We also quantified the cytokines in both fatal and non-fatal cases. For epidemiological correlation, livestock were screened for anti-CCHF IgG antibodies and the tick pool specimens were tested by real time RT-PCR. Virus isolation was attempted on tick pools and human specimens and phylogenetic analysis performed on human and ticks complete genome sequences. RESULTS: CCHF cases were detected throughout year in 2019 with the peak in August. Out of 34 cases, eight secondary CCHF cases were reported. Cases were predominantly detected in males and in 19-45 years age group (55.88%). The persistence of viremia was observed till 76th POD (post onset date) in one case whereas anti-CCHFV IgM and IgG was detected amongst these cases from the 2nd and 20th POD respectively. Positivity observed amongst livestock and tick pools were was 21.57% and 7.4% respectively. The cytokine analysis revealed a significant increase in the level of serum IL-6, IL-10 and IFN-γ during the acute phase of the infection, but interestingly IL-10 lowered to normal upon clearance of the virus in the clinically recovered case. Fatal cases had high viral RNA copy numbers. Bleeding from one or two mucosal sites was significantly associated with fatality (OR-16.47;p-0.0034 at 95% CI). We could do CCHF virus isolation from two cases. Phylogenetic analysis revealed circulation of re-assortment of Asian-West African genotypes in humans and ticks. CONCLUSIONS: The persistence of CCHF viral RNA was detected till 76th POD in one of the survivors. The circulation of a re-assortment Asian-West African genotype in a CCHF case is also reported first time from India.

Development of a Novel Multi-Epitope Vaccine Against Crimean-Congo Hemorrhagic Fever Virus: An Integrated Reverse Vaccinology, Vaccine Informatics and Biophysics Approach.

Crimean-Congo hemorrhagic fever (CCHF) is a highly severe and virulent viral disease of zoonotic origin, caused by a tick-born CCHF virus (CCHFV). The virus is endemic in many countries and has a mortality rate between 10% and 40%. As there is no licensed vaccine or therapeutic options available to treat CCHF, the present study was designed to focus on application of modern computational approaches to propose a multi-epitope vaccine (MEV) expressing antigenic determinants prioritized from the CCHFV genome. Integrated computational analyses revealed the presence of 9 immunodominant epitopes from Nucleoprotein (N), RNA dependent RNA polymerase (RdRp), Glycoprotein N (Gn/G2), and Glycoprotein C (Gc/G1). Together these epitopes were observed to cover 99.74% of the world populations. The epitopes demonstrated excellent binding affinity for the B- and T-cell reference set of alleles, the high antigenic potential, non-allergenic nature, excellent solubility, zero percent toxicity and interferon-gamma induction potential. The epitopes were engineered into an MEV through suitable linkers and adjuvating with an appropriate adjuvant molecule. The recombinant vaccine sequence revealed all favorable physicochemical properties allowing the ease of experimental analysis in vivo and in vitro. The vaccine 3D structure was established ab initio. Furthermore, the vaccine displayed excellent binding affinity for critical innate immune receptors: TLR2 (-14.33 kcal/mol) and TLR3 (-6.95 kcal/mol). Vaccine binding with these receptors was dynamically analyzed in terms of complex stability and interaction energetics. Finally, we speculate the vaccine sequence reported here has excellent potential to evoke protective and specific immune responses subject to evaluation of downstream experimental analysis.

Development of double antibody sandwich ELISA as potential diagnostic tool for rapid detection of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) virus, a highly pathogenic viral agent is responsible for severe fatal hemorrhagic infections in many parts of the world. The early diagnosis of CCHF infection is important for successful clinical management and epidemiological control. The nucleoprotein (NP) of CCHFV being highly conserved and immunogenic is used as early diagnostic marker. In this study, we report a rapid and sensitive double antibody based antigen capture ELISA to detect Crimean-Congo hemorrhagic fever virus (CCHFV). Highly specific polyclonal and monoclonal antibody against NP has been generated and used as capture and detector antibody respectively. The assay was able to detect viral nucleoprotein in different matrices including human serum, ticks and culture supernatant. The detection limit of the developed sandwich ELISA assay was 25 ng of purified antigen. Comparison with a real time RT-PCR revealed its detection limit to be 1000 genome equivalents of CCHFV. Further the assay was comparatively evaluated with a commercial kit employing gamma irradiated CCHFV, revealing a sensitivity and specificity of 100%. This newly developed sandwich ELISA (sELISA) with high sensitivity and specificity could be used as an efficient method for the detection of CCHF virus in humans, ticks and culture supernatant. The assay will be useful as alternate tool for diagnosis of acute infection and is amenable for screening of large scale samples in resource limited settings.

Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic.

Viral replicon particles protect IFNAR-/- mice against lethal Crimean-Congo hemorrhagic fever virus challenge three days after vaccination.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes mild to severe and fatal disease in humans. Person-to-person transmission is common, necessitating the availability of rapidly deliverable therapeutic and prophylactic interventions to mitigate CCHFV spread. Previously, we showed complete protection using one dose of a viral replicon particle (VRP) vaccine administered 28 days before CCHFV challenge. In order to determine the utility of the VRP vaccine for rapid vaccination protocols, we assessed the efficacy of such vaccination administered at various intervals relative to challenge in IFNAR-/- mice. Unvaccinated mice uniformly succumbed to disease by 8 days post infection (dpi). All mice vaccinated 14, 7, or 3 days prior to CCHFV challenge survived infection. Mice vaccinated -14 or -7 dpi were fully protected from clinical disease, whereas mice inoculated -3 dpi developed signs of disease prior to recovering to baseline values 5-9 dpi. These data support the utility of the VRP vaccine for modified short course vaccination protocols to protect against disease and severe outcomes.

Epidemiology of Crimean-Congo Hemorrhagic Fever (CCHF) in Africa-Underestimated for Decades.

Crimean-Congo hemorrhagic fever (CCHF) is endemic in Africa, but the epidemiology remains to be defined. Using a broad database search, we reviewed the literature to better define CCHF evidence in Africa. We used a One Health approach to define the impact of CCHF by reviewing case reports, human and animal serology, and records of CCHF virus (CCHFV) isolations (1956-mid-2020). In addition, published and unpublished collection data were used to estimate the geographic distribution of Hyalomma ticks and infection vectors. We implemented a previously proposed classification scheme for organizing countries into five categories by the level of evidence. From January 1, 1956 to July 25, 2020, 494 CCHF cases (115 lethal) were reported in Africa. Since 2000, nine countries (Kenya, Mali, Mozambique, Nigeria, Senegal, Sierra Leone, South Sudan, Sudan, and Tunisia) have reported their first CCHF cases. Nineteen countries reported CCHF cases and were assigned level 1 or level 2 based on maturity of their surveillance system. Thirty countries with evidence of CCHFV circulation in the absence of CCHF cases were assigned level 3 or level 4. Twelve countries for which no data were available were assigned level 5. The goal of this review is to inform international organizations, local governments, and healthcare professionals about shortcomings in CCHF surveillance in Africa to assist in a movement toward strengthening policy to improve CCHF surveillance.

Experimental Challenge of Sheep and Cattle with Dugbe Orthonairovirus, a Neglected African Arbovirus Distantly Related to CCHFV.

Dugbe orthonairovirus (DUGV) is a tick-borne arbovirus within the order Bunyavirales. DUGV was first isolated in Nigeria, but virus isolations in ten further African countries indicate that DUGV is widespread throughout Africa. Humans can suffer from a mild febrile illness, hence, DUGV is classified as a biosafety level (BSL) 3 agent. In contrast, no disease has been described in animals, albeit serological evidence exists that ruminants are common hosts and may play an important role in the transmission cycle of this neglected arbovirus. In this study, young sheep and calves were experimentally inoculated with DUGV in order to determine their susceptibility and to study the course of infection. Moreover, potential antibody cross-reactivities in currently available diagnostic assays for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) were assessed as DUGV is distantly related to CCHFV. Following subcutaneous inoculation, none of the animals developed clinical signs or viremia. However, all ruminants seroconverted, as demonstrated by two DUGV neutralization test formats (micro-virus neutralization test (mVNT), plaque reduction (PRNT)), by indirect immunofluorescence assays and in bovines by a newly developed DUGV recombinant N protein ELISA. Sera did not react in commercial CCHFV ELISAs, whereas cross-reactivities were observed by immunofluorescence and immunoblot assays.

Development of a novel recombinant ELISA for the detection of Crimean-Congo hemorrhagic fever virus IgG antibodies.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV). Serological screening of CCHF is important and current ELISA use antigens prepared from virus which is expensive due to requirement of high bio-containment facilities. In this study, we aimed to develop a new recombinant ELISA. For this purpose, CCHFV genome were expressed as 13 proteins in E. coli and among them abundantly purified recombinant Nucleocapsid protein (rNP) and Mucin-like variable domain (rMLD) were used as antigen in ELISA (Rec-ELISA). Rec-ELISA using rNP, rMLD and a combination of both (rNP/rMLD) were probed with acute (n = 64; collected between days 1 and 7 after onset of symptoms), convalescent (n = 35; collected 8 days after onset of symptoms), consecutive sera (n = 25) of confirmed CCHF cases and control sera (n = 43). The sensitivity and specificity of Rec-ELISA using rNP/rMLD were 73% and 98% in acute cases and 97% and 98% in convalescent cases. The median interquartile absorbance value to discriminate the acute and convalescent phases of CCHF was significantly higher with ELISA using rNP/rMLD (P < 0.0001) compared to rNP (P > 0.05) and rMLD (P = 0.001). These results indicate that the Rec-ELISA using rNP/rMLD may be very useful to diagnose convalescent CCHF cases especially in field studies.

Immunocompetent mouse model for Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne febrile illness with wide geographic distribution. CCHF is caused by infection with the Crimean-Congo hemorrhagic fever virus (CCHFV) and case fatality rates can be as high as 30%. Despite causing severe disease in humans, our understanding of the host and viral determinants of CCHFV pathogenesis are limited. A major limitation in the investigation of CCHF has been the lack of suitable small animal models. Wild-type mice are resistant to clinical isolates of CCHFV and consequently, mice must be deficient in type I interferon responses to study the more severe aspects of CCHFV. We report here a mouse-adapted variant of CCHFV that recapitulates in adult, immunocompetent mice the severe CCHF observed in humans. This mouse-adapted variant of CCHFV significantly improves our ability to study host and viral determinants of CCHFV-induced disease in a highly tractable mouse model.

Crimean-Congo Hemorrhagic Fever Virus Past Infections Are Associated with Two Innate Immune Response Candidate Genes in Dromedaries.

Dromedaries are an important livestock, used as beasts of burden and for meat and milk production. However, they can act as an intermediate source or vector for transmitting zoonotic viruses to humans, such as the Middle East respiratory syndrome coronavirus (MERS-CoV) or Crimean-Congo hemorrhagic fever virus (CCHFV). After several outbreaks of CCHFV in the Arabian Peninsula, recent studies have demonstrated that CCHFV is endemic in dromedaries and camel ticks in the United Arab Emirates (UAE). There is no apparent disease in dromedaries after the bite of infected ticks; in contrast, fever, myalgia, lymphadenopathy, and petechial hemorrhaging are common symptoms in humans, with a case fatality ratio of up to 40%. We used the in-solution hybridization capture of 100 annotated immune genes to genotype 121 dromedaries from the UAE tested for seropositivity to CCHFV. Through univariate linear regression analysis, we identified two candidate genes belonging to the innate immune system: FCAR and CLEC2B. These genes have important functions in the host defense against viral infections and in stimulating natural killer cells, respectively. This study opens doors for future research into immune defense mechanisms in an enzootic host against an important zoonotic disease.

Investigation of Crimean-Congo hemorrhagic fever virus in ruminant species slaughtered in several endemic provinces in Turkey.

A total of 1,337 serum and plasma specimens (939, 393 and 15 from cattle, sheep and goats, respectively) were collected monthly for one a year from ruminant species slaughtered in three Turkish cities endemic for Crimean-Congo hemorrhagic fever virus (CCHFV), Samsun, Sivas and Tokat. The serum samples were tested by commercial indirect ELISA to detect CCHFV antibodies, and positive or equivocal samples were later confirmed by a virus neutralization test (VNT). The seroprevalence in cattle, sheep, and goats was 36.21% (340/939), 6.27% (24/383), and 6.67% (1/15), respectively. Quantitative real-time RT-PCR was employed to detect viraemic animals at slaughter time. The percentage of CCHFV-viraemic animals was 0.67% (9/1337). The virus load varied between 4.1 x 101 and 2.4 x 103 RNA equivalent copies/mL in viraemic animals. The plasma samples that were positive for CCHFV genomic RNA were collected between April and May, when Hyalomma ticks are active. This study presents quantitative CCHFV load data in ruminant species at slaughter and interprets the likelihood of transmission for employees working in slaughterhouses in CCHFV-endemic regions.

Structure and Characterization of Crimean-Congo Hemorrhagic Fever Virus GP38.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a ß-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

Introducing of an integrated artificial neural network and Chou's pseudo amino acid composition approach for computational epitope-mapping of Crimean-Congo haemorrhagic fever virus antigens.

This study was aimed to introduce a novel algorithm for determining linear B- and T-cell epitopes from Crimean-Congo haemorrhagic fever virus (CCHFV) antigens. To this end, 387 approved B- and T-cell epitopes, as well as 331 non-epitope peptides from different serotypes of the virus were collected from IEDB database for generating of the train datasets. After that, the physicochemical properties of the epitopes were expressed as the numeric vectors using Chou's pseudo amino acid composition method. The vectors then were used for training of four machine learning algorithms including artificial neural network (ANN), k-nearest neighbors (kNN), support vector machine (SVM) and Random forest (RF). The results confirmed that ANN was the most accurate algorithm for discriminating between the epitopes and non-epitopes with the accuracy of 0.90. Furthermore, for evaluating the performance of the ANN algorithm, an epitope prediction challenge was performed to a random peptide library from envelopment polyprotein of CCHFV. Moreover, the efficiency of the predicted epitopes in term of antigenicity and affinity to MHC-II were compared to the predicted epitope by standard epitope prediction tools based on their VaxiJen 2.0 score and molecular docking outputs. Finally, the ability of the screened epitopes to stimulation of humoral and cellular responses was evaluated by an in silico immune simulation process thought C-Immsim 10.1 server. The results confirmed that this method has more accuracy for epitope-mapping than the standard tools and could considered as an effective algorithm to develop a serotype independent one-click automated epitope based vaccine design tool.

Fine mapping epitope on glycoprotein Gc from Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis, caused by CCHF virus (CCHFV) and which there are no diagnostic or therapeutic strategies. The C-terminus of glycoprotein (Gc) encoded by the CCHFV M gene is responsible for CCHFV binding to cellular receptors and acts as a neutralizing-antibody target. In this study, a modified biosynthetic peptide technique (BSP) was used to identify fine epitopes of Gc from the CCHFV YL04057 strain using rabbit antiserum against CCHFV-Gc. Six B cell epitopes (BCEs) and one antigenic peptide (AP) were identified: E1 (88VEDASES94), E2 (117GDRQVEE123), E3 (241EIVTLH246), AP-4 (281DFQVYHVGNLLRGDKV296), E5a (370GDTP QLDL377), E5b (373PQLDLKAR380), and E6 (443HVRSSD448). Western blotting analysis showed that each epitope interacted with the positive serum of sheep that had been naturally infected with CCHFV, and the results were consistent with that of Dot-ELISA. The multiple sequence alignment (MSA) revealed high conservation of the identified epitopes among ten CCHFV strains from different areas, except for epitopes AP-4 and E6. Furthermore, three-dimensional structural modeling showed that all identified epitopes were located on the surface of the Gc "head" domain. These mapped epitopes of the CCHFV Gc would provide a basis for further increase our understanding CCHFV glycoprotein function and the development of a CCHFV epitope-based diagnostics vaccine and detection antigen.

Seroepidemiological survey of Crimean-Congo haemorrhagic fever among high-risk groups in the west of Iran.

Crimean-Congo haemorrhagic fever (CCHF) is a zoonotic viral haemorrhagic disease. This disease is more common in people who work with animals infected with CCHF virus. The aim of this study was to evaluate the CCHF exposure in high-risk occupational groups in Kurdistan Province in the west of Iran. This cross-sectional study was conducted in 2014 in three counties of Kurdistan Province, viz. Sanandaj, Marivan and Sarvabad. About 50 butchers and slaughterhouse workers, 50 hunters, 50 health care workers and 100 subjects referred to clinical laboratories were sampled and examined for the diagnosis of IgG antibodies against the CCHF using ELISA method. The serum sample of one of the butchers and slaughterhouse workers was positive for CCHF virus. No positive case was found in any other studied groups. The study findings indicate that although CCHF is an endemic disease in different parts of Iran, there is a low rate of seropositivity among high-risk occupations in the west of Iran. Therefore, it is not probably a serious public health problem in this area.

Heterologous protection against Crimean-Congo hemorrhagic fever in mice after a single dose of replicon particle vaccine.

No vaccines are currently licensed to prevent Crimean-Congo hemorrhagic fever virus (CCHFV) infection, which can cause mild self-limiting clinical signs or severe, often fatal hemorrhagic fever disease. Here we continued investigations into the utility of a single-dose virus replicon particle (VRP) vaccine regimen by assessing protection against Turkey or Oman strains of CCHFV. We found that all mice were completely protected from disease, supporting broad applicability of this platform for CCHFV prevention.

The Role of Reference Materials in the Research and Development of Diagnostic Tools and Treatments for Haemorrhagic Fever Viruses.

Following the Ebola outbreak in Western Africa in 2013-16, a global effort has taken place for preparedness for future outbreaks. As part of this response, the development of vaccines, treatments and diagnostic tools has been accelerated, especially towards pathogens listed as likely to cause an epidemic and for which there are no current treatments. Several of the priority pathogens identified by the World Health Organisation are haemorrhagic fever viruses. This review provides information on the role of reference materials as an enabling tool for the development and evaluation of assays, and ultimately vaccines and treatments. The types of standards available are described, along with how they can be applied for assay harmonisation through calibration as a relative potency to a common arbitrary unitage system (WHO International Unit). This assures that assay metrology is accurate and robust. We describe reference materials that have been or are being developed for haemorrhagic fever viruses and consider the issues surrounding their production, particularly that of biosafety where the viruses require specialised containment facilities. Finally, we advocate the use of reference materials at early stages, including research and development, as this helps produce reliable assays and can smooth the path to regulatory approval.

GP38-targeting monoclonal antibodies protect adult mice against lethal Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. Limited evidence suggests that antibodies can protect humans against lethal CCHFV disease but the protective efficacy of antibodies has never been evaluated in adult animal models. Here, we used adult mice to investigate the protection provided against CCHFV infection by glycoprotein-targeting neutralizing and non-neutralizing monoclonal antibodies (mAbs). We identified a single non-neutralizing antibody (mAb-13G8) that protected adult type I interferon-deficient mice >90% when treatment was initiated before virus exposure and >60% when administered after virus exposure. Neutralizing antibodies known to protect neonatal mice from lethal CCHFV infection failed to confer protection regardless of immunoglobulin G subclass. The target of mAb-13G8 was identified as GP38, one of multiple proteolytically cleaved glycoproteins derived from the CCHFV glycoprotein precursor polyprotein. This study reveals GP38 as an important antibody target for limiting CCHFV pathogenesis and lays the foundation to develop immunotherapeutics against CCHFV in humans.